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Sporulation in Bacteria:
Beyond the Standard Model
ELIZABETH A. HUTCHISON,1
DAVID A. MILLER,2 and ESTHER R. ANGERT3
1
Department of Biology, SUNY Geneseo, Geneseo, NY 14454; 2Department of Microbiology,
Medical Instill Development, New Milford, CT 06776; 3Department of Microbiology,
Cornell University, Ithaca, NY 14853
ABSTRACT Endospore formation follows a complex, highly
regulated developmental pathway that occurs in a broad range
of Firmicutes. Although Bacillus subtilis has served as a powerful
model system to study the morphological, biochemical, and
genetic determinants of sporulation, fundamental aspects of the
program remain mysterious for other genera. For example,
it is entirely unknown how most lineages within the Firmicutes
regulate entry into sporulation. Additionally, little is known about
how the sporulation pathway has evolved novel spore forms and
reproductive schemes. Here, we describe endospore and
internal offspring development in diverse Firmicutes and outline
progress in characterizing these programs. Moreover,
comparative genomics studies are identifying highly conserved
sporulation genes, and predictions of sporulation potential in
new isolates and uncultured bacteria can be made from these
data. One surprising outcome of these comparative studies is
that core regulatory and some structural aspects of the program
appear to be universally conserved. This suggests that a robust
and sophisticated developmental framework was already in
place in the last common ancestor of all extant Firmicutes
that produce internal offspring or endospores. The study of
sporulation in model systems beyond B. subtilis will continue to
provide key information on the flexibility of the program and
provide insights into how changes in this developmental course
may confer advantages to cells in diverse environments.
AN INTRODUCTION TO
ENDOSPORE FORMATION
Bacteria thrive in amazingly diverse ecosystems and
often tolerate large fluctuations within a particular environment. One highly successful strategy that allows a
cell or population to escape life-threatening conditions
is the production of spores. Bacterial endospores, for
example, have been described as the most durable cells
in nature (1). These highly resistant, dormant cells can
withstand a variety of stresses, including exposure to
temperature extremes, DNA-damaging agents, and hydrolytic enzymes (2). The ability to form endospores
appears restricted to the Firmicutes (3), one of the earliest branching bacterial phyla (4). Endospore formation
is broadly distributed within the phylum. Spore-forming
species are represented in most classes, including the
Bacilli, the Clostridia, the Erysipelotrichi, and the Negativicutes (although compelling evidence to demote this
class has been presented [5]). To the best of our knowledge endospores have not been observed in members
of the Thermolithobacteria, a class that contains only a
few species that have been isolated and studied. Thus,
sporulation is likely an ancient trait, established early
in evolution but later lost in many lineages within the
Firmicutes (4, 6).
Endospores occur most commonly in rod-shaped bacteria (Fig. 1), but also appear in filamentous cells and in
cocci (7–11). Many endospores have been observed only
in samples from nature. For instance, large, morphologically diverse helical bacteria (40 to 100 μm long), named
Sporospirillum spp., produce one or two endospore-like
Received: 13 November 2012, Accepted: 27 August 2014,
Published: 3 October 2014
Editors: Patrick Eichenberger, New York University, New York, NY,
and Adam Driks, Loyola University Medical Center, Maywood, IL
Citation: Hutchison EA, Miller DA, Angert ER. 2014. Sporulation in
bacteria: beyond the standard model. Microbiol Spectrum
2(5):TBS-0013-2012. doi:10.1128/microbiolspec.TBS-0013-2012.
Correspondence: Esther R. Angert, [email protected]
© 2014 American Society for Microbiology. All rights reserved.
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Hutchison et al.
FIGURE 1 Bacteria that produce endospores or intracellular offspring exhibit a wide
variety of morphological phenotypes. (A) Phase-contrast microscopy is often used to
identify mature endospores (A to C and E) as these highly mineralized cells appear phasebright. In this image of B. subtilis, the caret (>) indicates a cell that is not dividing or
sporulating and the asterisk (*) indicates a cell undergoing binary fission. All other cells in
the image contain a phase-bright endospore. (B) Clostridium oceanicum frequently
produces phase-bright endospores at both ends of the cell. Image courtesy of Avigdor
Eldar and Michael Elowitz, California Institute of Technology. (C) In this image of Anaerobacter polyendosporus, the arrows indicate cells with seven endospores. (D) The fluorescence micrograph of Metabacterium polyspora outlines cell membranes and spore
coats stained with FM1-43. (E) Epulopiscium-like type C (cigar-shaped cell) and type J
(elongated cells), each containing two phase-bright endospores. (F) Epulopiscium sp.
type B with two internal daughter cells, stained with DAPI. Cellular DNA is located at the
periphery of the cytoplasm in the mother cell and each offspring. (G) Scanning electron
micrograph (SEM) of the ileum lining from a rat reveals the epithelial surface densely
populated with SFB. Arrow indicates a holdfast cell that has not yet elongated into a
filament. (H) Transmission electron micrograph (TEM) of a thin section through the gut
wall reveals the structure of the SFB holdfast cell (indicated by an asterisk). (I to J) TEMs
illustrate the two possible fates for developing intracellular SFB: (I) two holdfast cells or
(J) two endospores that are encased in a common coat (C), inner (I) and outer (O) cortex.
Panel C reproduced from Siunov et al. (47) with permission from Society for General
Microbiology. Panel E reproduced from Flint et al. (33) with permission from ASM Press.
Panel F reproduced from Mendell et al. (93) with permission from the National Academy
of Sciences, USA. Panels G and H reproduced from Erlandsen and Chase (69) with
permission from the American Society for Nutrition. Panels I and J reproduced from
Ferguson and Birch-Andersen (74) with permission from John Wiley and Sons. doi:10.1128
/microbiolspec.TBS-0013-2012.f1
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Sporulation in Bacteria: Beyond the Standard Model
structures (12, 13). These bacteria have been found in
the gut of batrachian tadpoles, although their affiliation
within the Firmicutes has not been established. The diversity of endospore-producing bacteria and their varied
lifestyles suggest that the sporulation pathway is finely
tuned to life in a particular environment, and is an advantageous means of cellular survival, dispersal, and, in
some cases, reproduction.
The basic and most familiar mode of sporulation
(Fig. 2A) involves an asymmetrical cell division that leads
to the formation of a mother cell and a smaller forespore
(14, 15). Unique transcriptional programs within these
cells result in distinct fates for the forespore and the
mother cell. The initiation of sporulation in Bacillus
subtilis is triggered by a lack of nutrients and by high cell
density (2, 15). The decision to sporulate is tightly regulated, because this energy-intensive process serves as
a last resort for these starving cells. In the early stages
of sporulation, gene regulation mainly depends on the
stationary-phase sigma factor σH and the master transcriptional regulator Spo0A (16, 17). Activation of
Spo0A in B. subtilis is governed by a phosphorelay system involving several kinases, each of which transmits
information about cell condition and environmental
stimuli to determine the phosphorylation state of the intracellular pool of Spo0A (18). Prior to asymmetric cell
division, the chromosome replicates, and each replication origin rapidly migrates to a different pole of the
cell (19). Subsequently, the origin-proximal regions become tethered to opposite poles and the chromosomal
DNA stretches from one pole to the other to form
an axial filament (20, 21). During division, only ∼30%
of the origin-proximal portion of one chromosome is
trapped within the forespore, and the rest is translocated
into the forespore by SpoIIIE, a DNA transporter protein
(17, 22). The other chromosome copy remains in the
mother cell.
Differential activation of sporulation-specific sigma
factors in the mother cell and forespore manages the fate
of each cell (14). First, σF is activated exclusively in the
forespore (17). Shortly thereafter, a signal is sent to the
mother cell to process and hence activate σE. Both early
sigma factors promote the expression of genes necessary
for forespore engulfment, as well as genes needed for the
production and activation of the late sporulation sigma
factors (17, 23). Remodeling of septal peptidoglycan
allows migration of the mother-cell membrane around
the forespore (2, 17, 24, 25). Eventually, the leading
edge of the migrating mother-cell membrane meets, and
fission establishes the double-membrane-bound forespore within the mother cell. Completion of forespore
FIGURE 2 Endospore development. In monosporic bacteria,
complete division occurs at only one end of the developing
sporangium (A), while bacteria that produce two endospores
generally divide at both poles (B). In some lineages, such as the
SFB and M. polyspora, engulfed forespores undergo division (not
shown). Note that at least three chromosome copies are required to produce two viable endospores. Following endospore
engulfment, cortex and coat layers develop, and upon endospore maturation, the mother cell lyses, releasing one (A) or two
(B) endospores. doi:10.1128/microbiolspec.TBS-0013-2012.f2
engulfment, combined with further intercellular signaling, allows activation of σG in the forespore and the
subsequent activation of σK in the mother cell. These
sigma factors regulate the genes necessary for spore
maturation and germination (2, 17). Ultimately, the
mother cell undergoes programmed cell death and lysis,
which releases the mature endospore (26, 27).
TWIN ENDOSPORE FORMATION
IN B. SUBTILIS AND TWINS
PRODUCED IN NATURE
Although sporogenesis in B. subtilis typically culminates in the production of a single endospore, simple
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Hutchison et al.
mutations can vary the outcome of the program and lead
to the production of two viable, mature spores (28). This
is due in part to the normal assembly of functional division apparati at both ends of the cell even if only one
is used. Null mutations that block activation or expression of σE will arrest sporulation after asymmetric division. These mutants produce abortive disporics where
the developing sporangium divides sequentially at both
poles and chromosome copies are transferred into each
of the polar forespores, leaving the mother cell devoid of
a chromosome. Forespore-specific expression of spoIIR
is necessary for intercellular signaling to activate σE in
the mother cell. Eldar et al. found that, by manipulating
the expression of spoIIR, a small percentage of cells
“escape” sporulation, resume chromosome replication,
and then undergo division at both poles to produce viable and UV-resistant “twin endospores.” When combined with mutations that increase chromosome copy
number, such as those that prevent expression of the
replication inhibitor yabA, the frequency of twins in the
population elevates, provided that the mother cell retains a copy of the chromosome (28). Data from Eldar
et al. and studies of other sporulation systems (discussed
later) suggest that natural mutations that increase ploidy
and promote bipolar division could gradually increase
the occurrence of this alternative developmental outcome, thus leading to twin endospore formation as a
means of reproduction (28, 29).
Several bacterial lineages naturally produce these
“fraternal” twin endospores. The marine anaerobe Clostridium oceanicum is a rod-shaped bacterium that typically produces two endospores (Fig. 1B), depending on
the temperature or medium composition (30). The DNA
replication and septation events (Fig. 2B) leading to
twin endospore formation in C. oceanicum closely resemble those of twin endospore formation in B. subtilis
in the mutant strains described above (28). Although
rare, twin endospores naturally occur in Bacillus thuringiensis as well (31). Other twin endosporeformers, such
as the large, rod-shaped spore-forming bacteria from the
intestinal tract of batrachian tadpoles and rodents (12,
13, 32, 33), have been observed, but many of these have
not been phylogenetically characterized. Finally, the regular production of twin endospores has been described
in Epulopiscium-like cells (34). Twin endospore-forming
bacteria are frequently observed in the gastrointestinal
tract, which suggests that these nutrient-rich ecosystems
may better support increased ploidy (35), a requisite for
the production of more than one endospore.
Epulopiscium spp. and their close relatives, known
as “epulos,” are intestinal inhabitants of certain species
of surgeonfish (36). All morphotypes characterized
to date are exceptionally large, with some reaching
600 μm (37–39). Due to their large size, Epulopiscium
spp. were originally classified as protists (36, 39–41),
but further ultrastructural and molecular phylogenetic
analyses proved that these symbionts are bacteria (37,
38). Phylogenetically, epulos group within the clostridial cluster XIVb in the Lachnospiraceae (34, 37, 42, 43).
A survey of surgeonfish intestinal communities provided
a first assessment of the distribution of epulos among
host species and classified these diverse symbionts into
ten morphotypes (A to J) based on their cellular and
reproductive characteristics (36).
These surgeonfish symbionts exhibit a variety of
novel reproductive patterns (44). Only the two largest
morphotypes, A and B, are referred to as Epulopiscium
spp., and these lineages appear to reproduce solely by
the formation of multiple, nondormant intracellular
offspring (39, 40, 45), which will be described below.
Some of the smaller epulo morphotypes undergo binary
fission, and many have the ability to produce phasebright endospores (34, 36, 39, 40, 45). The generation
of intracellular offspring in Epulopiscium spp. or of
endospores in smaller epulo morphotypes is similar to
endosporulation in other Firmicutes, and developmental
progression can be highly synchronized in naturally occurring populations (34, 39, 45, 46).
The phase-bright endospores of the epulo C and J
morphotypes from the surgeonfish Naso lituratus have
been described in the most detail (34). Type C cells are
typically cigar shaped, 40 to 130 μm long, and do not
undergo binary fission, while type J cells are thin filaments, 40 to 400 μm long, and capable of binary fission
(Fig. 1E). Endospore maturation in type C and type
J epulos occurs nocturnally in a highly synchronized
manner, with 95 to 100% of the cells in these populations producing spores. Endospores are not seen in fish
during daylight hours, suggesting that epulos have
evolved a mechanism to regulate endospore development and germination in a diurnal fashion (34). Formation of endospores may promote offspring survival
by entering a period of dormancy when nutrients in
the gut become depleted, as the host fish sleeps. These
endospores may be more resilient than an actively
growing epulo, and could aid in transfer to a new host,
although the importance of spores in transmission has
yet to be fully evaluated (34). Thus, type C and J epulos
have modified their sporulation program to produce
“fraternal” twin endospores and coordinate this developmental program with regular fluctuations in their
environment.
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Sporulation in Bacteria: Beyond the Standard Model
SPORULATION PROGRAMS THAT CAN
PRODUCE MORE THAN TWO ENDOSPORES
While the bacteria discussed above have the ability to
form twin endospores, others have evolved the means
of producing more than two endospores per cell. Endospore formation is generally considered a survival
strategy, but the study of these multiple endosporeforming bacteria could provide insight into its use as a
reproductive strategy, which may be better suited to
some bacterial lifestyles than binary fission alone.
Anaerobacter polyendosporus was first isolated in
1985 from rice paddy soil (47). Depending on growth
conditions, A. polyendosporus can produce up to seven
endospores per cell (Fig. 1C). Cultures of A. polyendosporus are pleomorphic (47, 48), and the varying cell
types appear related to the metabolic transitions that
lead cells to sporulate, although this has not been the
subject of targeted studies. Thick rods with rounded
ends predominate cultures in exponential growth. Cells
become wider as the culture ages and eventually thick,
phase-bright rods and football-shaped cells appear. All
of these forms undergo binary fission (A. M. Johnson and
E. R. Angert, unpublished data). Each football-shaped
sporangium generally produces one or two endospores.
Under certain conditions, such as growth on potato agar
or in a liquid medium containing galactose, cells with more
than two endospores are observed (47). Twin endospores
are produced by division at both cell poles (Johnson and
Angert, unpublished). Since A. polyendosporus is a member of the cluster I clostridia (43), some of the cell forms
observed in sporulating cultures may be homologous to
the phase-bright, spindle-shaped clostridial form observed
in others of this group such as Clostridium acetobutylicum
and Clostridium perfringens (49, 50). Little research has
been conducted on A. polyendosporus, and many questions remain regarding its ability to produce multiple
endospores, including the role of morphological transitions in sporulation and the factors that lead to the production of more than two endospores.
Metabacterium polyspora (Fig. 1D), an inhabitant of
the intestinal tract of guinea pigs, has been studied in
some detail, revealing insights into how and why these
cells produce multiple endospores (29). Cells of M. polyspora pass through the digestive system and rely on the
coprophagous character of guinea pigs for cycling back
into its original host and for transmission to new hosts
(51). The M. polyspora cell would not last long outside
the host, and only mature endospores appear to survive
transit through the mouth and stomach. Germination
occurs after the passage of spores into the small intestine.
While these cells have the ability to reproduce by binary
fission, not all cells use this process. In those that do,
binary fission occurs during a short period of time following germination (Fig. 3). After germination, the cells
quickly transition to sporulation. In fact, cells with polar
septa are often observed emerging from the soon-to-be
discarded spore coat. The production of multiple endospores, up to nine per cell (52), allows M. polyspora to
produce offspring that are prepared for conditions outside the host (29, 51). Considering the rapid passage of
material through the gut and possibly the limited time
M. polyspora spends inside the host, we speculate that
reproduction by the instant formation of multiple endospores is advantageous to the symbiotic lifestyle of
M. polyspora and has allowed it to move away from a
reliance on binary fission.
FIGURE 3 Life cycle of Metabacterium polyspora.
Endospores germinate (A) and, during outgrowth, a
cell may undergo binary fission (B) or immediately
begin to sporulate by dividing at the poles (C). The
forespores are engulfed (D), and the forespores may
undergo binary fission to produce additional forespores (E). Forespores then elongate (F) and develop
into mature endospores (G). Figure reproduced from
Ward and Angert (52) with permission from John
Wiley and Sons. doi:10.1128/microbiolspec.TBS-0013
-2012.f3
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To produce multiple endospores, cells of M. polyspora, like the twin endosporeformers described above,
divide at both poles (Fig. 3) (17, 51). Each of the forespores receives at least one copy of the chromosome
and another copy (or copies) is retained in the mother
cell (53). Normally, in B. subtilis, DNA replication
occurs once, early in sporulation, and any additional
rounds of initiation are inhibited during sporulation
(54, 55). In contrast, DNA replication in M. polyspora
occurs throughout development, even after forespore
engulfment (53). To form more than two spores, the
fully engulfed forespore(s) divide (51). Additionally,
DNA replication within forespores loads the endospores
with multiple chromosomes, allowing cells to enter
sporulation immediately after germination without the
requisite of binary fission or chromosome replication
seen in B. subtilis. Bacteria that have the ability to produce two or more endospores, like M. polyspora, have
been reported in other coprophagous rodents (33). For
intestinal symbionts, it appears that spore formation not
only provides protection from the harsh external environment and to the host’s natural barriers to infection,
but also the process may be modified to provide a consistent means of cellular propagation.
MODIFICATION OF THE SPORULATION
PROGRAM FOR PRODUCTION OF
NONDORMANT INTERNAL OFFSPRING
In some groups of bacteria, the sporulation program
has evolved to produce multiple intracellular offspring,
some of which no longer go through a dormancy period.
Notably, members of the genus Candidatus Arthromitus,
as well as members of the segmented filamentous bacteria
(SFB) (also known as Candidatus Savagella), reproduce
via filament segmentation and internal daughter cell
production in addition to forming endospores (56).
Candidatus Arthromitus and the SFB are Gram-positive,
sometimes motile, endospore-forming bacteria found
in the intestinal tract of a diverse array of organisms,
ranging from mammals, to birds, to fish, to arthropods
(56–60). The genus “Arthromitus” was first described
and characterized by Joseph Leidy in the mid-1800s
from his observations of filamentous bacteria in arthropods and other animals (61, 62). Phylogenetic analyses
revealed that SFB (from rats, mice, chickens, and fish)
form a distinct clade within the group I clostridia, while
spore-forming filaments from arthropods constitute a
distinct group within the Lachnospiraceae (56–59, 63–
66). Isolates from different host species are distinct (56)
and exhibit host specificity (67, 68).
As yet, none of the SFB from vertebrate hosts are
available in pure culture, although the development of
gnotobiotic mammalian hosts mono-associated with SFB
has been successful for some lineages (69). Genome sequences derived from populations established in rodents
revealed that these bacteria lack almost all biosynthetic
pathways for amino acids, vitamins, cofactors, and nucleotides (63–65). The SFB likely live off simple sugars
and other essential nutrients gleaned from the host and
surrounding environment.
SFB can be abundant in the mammalian host (Fig. 1G)
but are restricted to the distal ileum (70, 71). SFB filaments are predominantly attached to the ileal wall and
localized to the Peyer’s patches, specialized lymphoid
follicles that function in antigen sampling and surveillance in the small intestine (72, 73). Close examination
of the gut environment revealed that SFB are simultaneously present in various stages of their life cycle,
including unattached teardrop-shaped cells in the intervillar spaces, and long or short filaments attached to
the ileal epithelium (71). The conical tip of the teardropshaped cell is referred to as the holdfast, which anchors
the cell to the epithelium. Upon attachment of a holdfast
cell, distinct morphological changes occur. The conical
tip of the holdfast protrudes into, but does not penetrate,
the membrane of the host epithelial cell (Fig. 1H) (70,
71, 74–76). In the host cell cytoplasm, the area adjacent
to the SFB attachment site forms an electron-dense layer
that comprises predominantly actin filaments (70, 77).
Although some holdfast cells appear to be phagocytosed
by the host, inflammation of the epithelial tissue at the
attachment site does not occur (78).
Once attached, the holdfast cell begins to elongate
and septate (Fig. 4A). SFB filaments are typically 50 to
80 μm, but can reach lengths up to 1,000 μm (70, 73).
As a filament transitions into its developmental cycle,
starting at the free end of the filament, the so-called
primary cells of the filament divide symmetrically, producing two equivalent secondary segments (Fig. 4A, iii)
(71, 75). These divisions establish an alternating orientation of cells in the filament, with respect to new and old
cell poles, which in turn appears to dictate the pattern
of asymmetric division of the secondary segments. After
secondary segment division, the larger cell engulfs the
smaller cell, which eventually forms a spherical body
within the larger mother cell (Fig. 4A, iv). These events
closely resemble the early stages of endospore formation
(71, 75, 76). Within each SFB mother cell, the engulfed
spherical body divides by first becoming crescent-shaped
(Fig. 4A, v to vi) and then constricting at the midcell,
leaving a pair of cells in each mother cell (71, 73, 75).
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Sporulation in Bacteria: Beyond the Standard Model
FIGURE 4 Life cycle of SFB and Epulopiscium sp. type B. (A) (i) The SFB life cycle begins
with a holdfast cell that is anchored to the
intestinal epithelia (not shown). (ii) Holdfast
cells elongate and divide into primary segments as the filament grows. (iii) At the start
of development, cells in the filament divide
again to produce secondary segments. (iv)
Next, secondary segments divide asymmetrically, and then engulfment of the smaller
cell (in grey) occurs, in a manner similar to
that of other endosporeformers. Development progresses from the free end of the
filament toward the holdfast. (v) Each engulfed offspring cell then forms into a crescent shape (vi) and then divides to either
form two holdfast offspring cells per segment (inset, top) or develop into an endospore via formation of a spore cortex and
coat (inset, bottom). (B) (i) In Epulopiscium
sp. type B, twin offspring form by division at
both cell poles. Engulfment occurs (ii to iii)
and offspring cells elongate (iv). The offspring cells begin to produce their own offspring before they are released from the
mother cell (v). doi:10.1128/microbiolspec
.TBS-0013-2012.f4
These “identical” twin offspring cells then differentiate
into holdfasts. At this point, the two cells can follow
one of two developmental pathways (Fig. 1I to J). In one,
the cells can progress through sporulation, producing a
cortex and two distinct coat layers. The emergent spores
are encased in a common spore coat, a feature that appears to be unique to the SFB sporogenesis pathway.
Eventually the mother cell deteriorates, releasing the
spore carrying these two offspring. Alternatively, the
holdfast cells are simply released upon mother cell lysis
(71, 75). A free holdfast cell may establish a new filament within the host, while the spore is an effective
dispersal vehicle capable of airborne infection of a naive
host (73). Thus, SFB have modified their developmental
program such that they can either produce two daughter
holdfast cells or an endospore that contains two cells,
likely conferring an advantage to this organism in the
dynamic environment of the gut and outside the host. It
is unclear how these alternative developmental processes
are instigated in a given filament or how different proportions of active or dormant cells impact population
dynamics.
The genetics of sporulation have not yet been characterized in detail for SFB, but genome sequence data
from these organisms suggest that many components
of the sporulation pathway from B. subtilis and clostridial genomes are conserved. Approximately 60 to 70
putative sporulation genes have been identified in SFB
genomes, including those coding for sporulation sigma
factors, stage-specific transcriptional regulators, and
spore germination proteins (63–65). Characterization
of the kinases that influence phosphorylation of Spo0A
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Hutchison et al.
could provide insight into factors that control the decision to sporulate or produce daughter cells, but, like
other clostridia, genes encoding the phosphorelay proteins in B. subtilis are absent in SFB (63, 64).
SFB have been adopted as a model for examining the
effect of commensals on host immune system development
and homeostasis. SFB have a broad range of immunostimulatory effects (79–82), and it has been suggested that
SFB affect pathogen resistance and autoimmune disease
susceptibility of their host (83–89). SFB are generally
considered harmless to a healthy host, and may provide
critical signals for immune development (90). However,
because of their intimate association with host cells and
potential to trigger an inflammatory response, the SFB
may contribute to disease susceptibility, depending on the
genetic background of the host and composition of its
resident gut microbiota (83, 91).
As an aside, sporulation in a group of unattached,
multicellular filamentous gut symbionts has been described in some morphological detail. Oscillospira guilliermondi, later called Oscillospira guilliermondii, is a
Gram-positive gastrointestinal bacterium found in the
cecum of guinea pigs and in the rumen of cattle, sheep,
and reindeer (92, 93). These filaments or ovals (5 to 100
μm long) are composed of a stack of disc-shaped cells that
in some ways resemble Beggiatoa spp., but are members
of the Ruminococcaceae or clostridial cluster IV (92–94).
Within a filament of Oscillospira, one or more sections
may produce an endospore. While nothing is known
about the genetics of sporulation in Oscillospira, ultrastructural images of filaments undergoing development
have been published, and the process appears to have
many of the hallmarks of endosporulation, including
forespore engulfment and the production of a spore with
a multilayered envelope of cortex and coat. Genome sequence from a non-spore-forming, closely related bacterium, Oscillibacter valericigenes, isolated from the gut
of a clam, revealed some conservation of sporulation
genes, particularly those involved in regulating early
events (95, 96).
As with the SFB described above, Epulopiscium spp.
type A and type B live successfully in the gut of a vertebrate host and exhibit an intracellular offspring developmental program. This process has been best studied in
type B cells from the host fish Naso tonganus (36, 45).
Epulopiscium sp. type B cells are very large, usually 100
to 300 μm long (Fig. 1F). Although some epulos, such
as types C and J, reproduce via endospore production
and/or binary fission, type B cells have never been observed to form endospores or undergo binary fission.
Instead, Epulopiscium sp. type B typically produces 2 to
3 nondormant, intracellular offspring per mother cell;
however, as many as 12 have been observed (29, 36).
To form offspring (Fig. 4B), Epulopiscium sp. type B
cells undergo asymmetric cell division, much like that
observed in classical endospore formation, but division
occurs at both cell poles (45). A given type B cell contains tens of thousands of copies of its genome to accommodate its large size, and polar division traps only
a small amount (<1%) of this DNA (45, 97). Next, the
insipient offspring are engulfed and grow within the
mother cell. Unlike endospore formation in B. subtilis,
DNA replication continues in both the mother cell and
offspring as the offspring grow (53). Upon completion
of offspring growth, the mother cell undergoes a form
of programmed cell death (45, 98). The entire developmental process occurs synchronously within a population. Given the close phylogenetic relationship of
Epulopiscium sp. type B and other epulos to endosporeforming bacteria, as well as the morphological similarities in the early stages of daughter cell development
to that of the early stages of endospore formation, it is
likely that the ancestor of all epulos produced endospores, and, with time, the program was modified to
function in intracellular offspring production in these
viviparous Firmicutes (42).
The Epulopiscium sp. type B genome has homologs of
the B. subtilis spoIIE gene and the spoIIA operon, which
contains genes coding for σF and its regulators SpoIIAA
and SpoIIAB (46). During sporulation in B. subtilis,
SpoIIE has dual roles: the promotion of asymmetric cell
division and the activation of σF. The pattern of spoIIE
expression with respect to asymmetric division and offspring development in Epulopiscium sp. type B populations is similar to that of B. subtilis, except that spoIIE
expression peaks slightly later in B. subtilis and stays
elevated for a longer developmental interval. Differences
in expression of spoIIE could be a consequence of
differences in the role of SpoIIE in each organism. Also,
it may reflect differences in population heterogeneity
because endospore formation is a last resort in B. subtilis
and cells delay entry into sporulation as long as possible
(99, 100), while development in Epulopiscium is essential for reproduction.
Epulopiscium sp. type B has become a model for
studies of cytoarchitecture and evolutionary potential.
These massive microbes are extremely polyploid and
maintain tens of thousands of genome copies throughout their life cycle (97). This adaptation appears essential for maintaining an active metabolism to support
such a large cytoplasmic volume (35). Likewise, polyploidy naturally provides one of the prerequisites of
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Sporulation in Bacteria: Beyond the Standard Model
multiple internal offspring production. Studies of the
Epulopiscium genome have revealed a tolerance for
unstable genetic elements, which appears to be a feature
shared with other polyploid symbionts (101). For
Epulopiscium specifically, extreme polyploidy and the
use of an endosporulation-derived reproduction have
led to the establishment of a cell with chromosomes of
differing fates (98). A small subset of chromosomes is
inherited by offspring directly, and we consider these
“germ line” chromosomes. Most chromosomal copies
remain in the mother cell after offspring are formed, and,
surprisingly, these chromosomes continue to replicate,
despite the fact that they cannot be directly passed on to
offspring. This suggests that replication of “somatic”
chromosomes is necessary to support the metabolic
needs of the mother cell and its growing offspring (98).
Studies of this unconventional bacterium are providing
fundamental insights into cellular biology and maintenance of genomic resources.
INSIGHTS FROM OTHER UNUSUAL
NONMODEL ENDOSPOREFORMERS
Thus far, we have focused on modifications of the basic
sporulation program to allow for the formation of
multiple endospores or multiple nondormant, intracellular offspring. Here, we describe two other noteworthy
and fruitful experimental systems that produce a single
endospore per mother cell.
Pasteuria spp., parasites of nematodes and Daphnia,
constitute another diverse group within the Firmicutes
that forms endospores that function in a remarkable
manner. Endospores of Pasteuria spp. consist of a spherical, opaque structure with several spore coat layers, and
an additional exosporial fibrillar matrix layer that skirts
the spore (102–105). This fibrillar matrix serves in hostspecific attachment. The attached Pasteuria spore germinates and produces a germ tube that enters the host,
where this obligate parasite grows and proliferates (102,
105). Sporogenesis of Pasteuria spp. has been characterized in microscopic detail, and a phylogenetic assessment
of these members of the Bacilli has been carried out
for the spo0A gene (106), yet the biology behind these
unique spore structures and factors that regulate germination and host specificity have yet to be characterized
fully. With the recent development of in vitro culturing
methods by Syngenta and Pasteuria Bioscience, Inc.,
the structure-function relationship of this unusual sporedelivery system may soon be uncovered.
Although the term Firmicutes is thought of as synonymous with “low G+C Gram-positive bacteria,” some
members of the family Veillonellaceae have a Gramnegative cell envelope and can form endospores. Recently, the process of sporulation was characterized in
stunning ultrastructural detail in one of these Gramnegative sporeformers, Acetonema longum (107). Using
3D electron cryotomographic imaging and immunodetection methods, Tocheva and colleagues show that,
through engulfment, the inner and outer membranes
of the A. longum mother cell become inverted. During
outgrowth, the membrane that was previously part of
the cytoplasmic membrane transforms, as outer membrane components such as lipopolysaccharide and
porins assemble in this now-exposed surface of the cell
envelope. The authors suggest that A. longum may provide insight into the mechanisms by which an outer
membrane could evolve, thus providing a plausible link
between early Gram-positive cell forms and the appearance of the Gram-negative envelope (107). Further,
this analysis provided evidence to support a hypothesis concerning peptidoglycan dynamics in all endosporeformers. When the state of peptidoglycan of the
developing spore was investigated, the authors found
that, during engulfment, a thin layer of peptidoglycan is
formed and this eventually becomes part of the Gramnegative periplasm (107). While analyses of this unusual
Gram-negative endospore-forming bacterium aimed at
elucidating unique features of this cell, its study provided additional evidence supporting a novel model of
peptidoglycan remodeling in driving a key forespore
developmental process, which was later confirmed in
B. subtilis (107).
EVOLUTION OF SPORULATION FROM A
COMPARATIVE GENOMICS PERSPECTIVE
Morphological comparisons between different species
and early genetic work on sporulation suggested that this
developmental pathway evolved only once in bacteria
(6, 108–110). As complete genome sequences became
available, comparative studies to look for conserved
sporulation genes became feasible (108, 111–114). In
one of the first extensive published surveys, Onyenwoke
et al. queried a set of 52 bacterial and archaeal genomes
using BLAST for 65 select B. subtilis sporulation genes
covering all stages of sporulation (108). Genes were
deemed part of the “core” sporulation pathway if they
were absent in non-spore-forming lineages but present
only in sporeformers or asporogenous strains (which
have conserved sporulation genes but do not produce
spores). With this approach, Onyenwoke et al. identified
a set of 45 sporulation-specific genes (108). In addition,
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Hutchison et al.
they noted differences between sporulation gene content
in Clostridia versus Bacilli genomes, and difficulties in
accurately identifying clostridial sporulation genes using
sequences from B. subtilis.
More recently, de Hoon et al. assessed the distribution of 307 B. subtilis genes that are directly regulated by
the sigma factors σH, σF, σE, σG, and σK in 24 different
species of spore-forming bacteria, using BLAST (6). The
authors confirmed that genes coding for the master
regulator of sporulation, spo0A, and the main sporulation sigma factors are conserved in all sporeformers
examined. Genes involved in signaling between sporulation sigma factors are also well conserved, but those
genes downstream in the signaling pathway (those that
function in a nonregulatory capacity) are not as conserved among sporeformers.
In an effort to improve the annotation of sporulation
genes and the ability to predict sporeformers from genomic data, Galperin et al. used a clusters of orthologous genes (COG)-based approach to identify a core
set of sporulation genes (96). The authors analyzed almost 400 Firmicutes genomes and sorted them into
spore-forming and non-spore-forming based on the
presence of spo0A, sspA, and dpaAB genes, which were
previously known to be fairly accurate predictors of
sporulation (108). The authors then compiled a list of
651 known sporulation genes and compared their distribution in sporeformers versus asporogenous strains
versus nonsporeformers. The authors presented a set of
approximately 60 genes conserved in members of the
Bacilli and Clostridia. Consistent with the idea that these
60 genes represent the minimum gene content for spore
formation, the sporulation gene complement in SFB genomes (which were published after the comparative
analysis by Galperin et al.) matches the predicted core
set almost exactly. SFB genomes are quite small (1.5 to
1.6 Mb) and appear streamlined (63–65, 115); therefore,
the SFB may represent a minimal, yet fully functional,
sporulation program. Abecasis et al. used a bidirectional
BLAST approach to identify 111 genes conserved in 90%
of known sporeformers (116). The authors refined this
further to a sporulation signature comprising 48 genes
that they used to predict sporulation competency. With
comparative genomics, the authors were able to distinguish bacteria that appeared to have recently lost the
ability to sporulate. In addition, they identified 22 species
that have not been observed to sporulate in culture, but
yet appear to have the ability to sporulate based on the
presence of complete sporulation signatures.
Another general finding of these studies is that some
members of the Firmicutes have retained many sporu-
lation genes despite their apparent inability to form
an endospore. As discussed previously in this review,
Epulopiscium sp. type B forms multiple intracellular
offspring cells using a process that is morphologically
similar to sporulation. A recent study by Miller et al.
used a BLAST-based approach to define and then compare the distribution of 147 highly conserved core
sporulation genes in Epulopiscium sp. type B as well
as the genome of its closest endospore-forming relative,
Cellulosilyticum lentocellum (117). While the C. lentocellum genome contains 87 of the core genes, the Epulopiscium sp. type B genome contains 57. The conserved
genes include homologs of spo0A, all sporulation sigma
factors, and the central regulatory network that governs
cell-specific transcriptional programs, as well as genes
required for engulfment. Late-stage sporulation genes
that confer resistance properties, such as the synthesis
and forespore transport of dipicolinic acid and germinant receptors located in the C. lentocellum genome,
were not found in Epulopiscium sp. type B. Surprisingly,
genes that code for small acid-soluble proteins (SASPs)
and their degradation, as well as cortex biosynthesis and
cortex/coat scaffolds, were conserved in both C. lentocellum and Epulopiscium sp. type B. It appears that
some of these late-stage functions may still be important
for Epulopiscium. Since endospores have never been
observed in Epulopiscium sp. type B, it is possible that
the conserved cortex-associated genes may provide a
specialized envelope to support the development and
rapid growth of daughter cells. SASPs may be important
for DNA protection or chromosome organization in developing offspring.
In general, comparative studies have confirmed that
the regulatory kinase cascade upstream of Spo0A is
not conserved (108), particularly not between Bacilli
and Clostridia. However, Spo0A and the sporulation
sigma factors (σH, σF, σE, σG, and σK) are universally
conserved in sporeformers. In addition, regulators of
these sigma factors, for example, spoIIAA, spoIIAB,
and the spoIIIA operon, are conserved. This suggests
that, despite the ways in which the sporulation pathway
has diverged among different Firmicutes lineages, these
core regulatory components are ancient and essential
for development. Previous morphological observations
suggested that engulfment, whether it is of a developing
forespore or a nondormant offspring cell, proceeds in
a very similar manner to that of B. subtilis, and indeed
genes involved in engulfment, such as spoIID, spoIIP,
spoIIM, and spoIIIE, are highly conserved among sporeformers. Finally, genes involved in spore coat production and germination are not well conserved among
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Sporulation in Bacteria: Beyond the Standard Model
endospore-forming bacteria, but this is not surprising
given the size of some of these proteins and the wide
range of environments in which sporeformers grow,
sporulate, and germinate.
An additional outcome of these comparative genomics studies is the finding that asporogenous and
nonsporeformers retain homologs to sporulation genes.
As more of these strains are characterized with respect
to sporulation, it will be interesting to see if these genes
have retained functions similar to that of their sporulation homologs or if they have become functionally divergent. Among the nonmodel sporeformers, there are
several species that can form more than two spores.
Since much of the engulfment machinery is conserved,
it is likely that these bacteria have found ways to either
engulf forespores that then divide to produce multiple
endospores (like M. polyspora), or to engulf at cellular
locations other than at the poles, as sometimes occurs
in Epulopiscium sp. type B cells (118). In the latter case,
it is currently unknown how these cells regulate where,
and how many, additional engulfment sites will occur.
Comparative genomics approaches have provided a valuable framework with which to assess the potential to
form a spore, and future work on nonmodel sporeforming organisms will provide insight into how sporulation genes evolve to function in diverse forms of
bacterial reproduction and development.
THE VALUE OF
COMPARATIVE APPROACHES
The sporulation pathway, as it has been classically
characterized, results in a single, stress-resistant spore
that allows a bacterium to survive unfavorable or even
potentially lethal environmental conditions. However,
bacteria have evolved and co-opted this pathway to
produce a wide range of endospore phenotypes, including multiple endospores and nondormant intracellular offspring. Although it is clear that forming an
endospore is advantageous for the survival of organisms
in harsh environments, the environmental or developmental triggers that control endospore production in
these more highly derived systems remain to be characterized fully. Of particular interest is how the production
of more than two endospores in some bacteria, such as
M. polyspora and A. polyendosporus, is regulated, especially since the number of spores produced varies
within populations of cells. Furthermore, the nuances
of why and how some bacteria alternate between multiple endospores or nondormant offspring have yet to be
fully elucidated.
A common theme presented here is that many of these
unusual developmental systems have been identified in
anaerobic, gastrointestinal symbionts. Our work, for
example, uses a comparative approach with closely related symbionts, and we have found that these systems
provide informative contrasts when considering the
impact of host-symbiont relations on the evolution of
novel reproductive strategies (29, 34, 42). All of these
intestinal symbionts are rather distant relatives of the
B. subtilis model, and we know that Clostridium spp.
use very different signals to trigger the onset of sporulation (109). Recent work on members of the Clostridia has reinforced previous observations that, while
sporulation genes are conserved between Clostridia and
Bacilli, frequently the regulation of these genes (including key sigma factors and their regulons) is different
between these two groups of sporeformers (119–121).
For example, in B. subtilis, σK functions exclusively late
in the sporulation pathway; however, in C. botulinum
(122) and C. perfringens (123), σK is required early in
sporulation. In C. acetobutylicum, σK is active both early
and late in development (124). In C. difficile, σK only has
a late role in sporulation, and a sigK mutant in C. difficile can be oligosporogenous (119, 121). Together,
these observations illustrate that the clean, sequential
model of sigma factor activation described for B. subtilis
does not fully represent patterns seen in the Clostridia
(119–121). We would suggest that the deep analysis of
additional spore-forming anaerobes, including genomic
and transcriptomic data, would provide a more robust
comparative system for generating hypotheses on triggers and modifications of the basic sporulation program.
The advent of high-throughput sequencing methods
has greatly expanded the ability to characterize uncultured bacteria, novel isolates with no established system for genetic dissection, and mutations that affect
development. Efforts to sequence diverse bacterial genomes are providing key insights into the conservation
of genes involved in sporogenesis (6, 108). In addition,
the application of high-resolution microscopy, including
fluorescence and cryotomographic imaging, is providing
unprecedented access to the cellular structures and processes associated with developmental progression. The
application of transcriptomics, proteomics, and comparative genomics to these unconventional systems will provide insight into the initiation process and potentially
identify triggers that determine alternative cell fates. Together, these efforts will provide a better understanding of
the conditions that repurpose sporulation, as well as the
potential diversity of form and function accommodated
by this complex and ancient developmental program.
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Hutchison et al.
ACKNOWLEDGMENTS
We thank Avigdor Eldar and Michael Elowitz from California
Institute of Technology for providing the image of C. oceanicum
and David Sannino, Jen Fownes, and Francine Arroyo for their
comments on this manuscript. We are also grateful to colleagues
who work with these and other unconventional model systems for
their insight.
Research in the Angert laboratory is supported by National
Science Foundation grants 0721583 and 1244378.
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